Fanconi anemia (FA) is a genetic disease with defects in development and hematopoiesis and propensity to cancer, indicating a vital and basic cell biology process at work. The hallmark of FA is genomic instability, evidenced by gross chromosomal breakage and DNA alkylating agent hypersensitivity, which correlates strongly with cancer predilection in general. Studies of FA are important in several ways. First, FA biology is involved across a spectrum of scientific disciplines, including hematology, oncology, and development. Second, since the known FA proteins are found only in mammalian cells and have no previously described protein domain, their study will yield the description of a novel pathway which promotes the maintenance of gnomic stability. Third, work on other proteins derived from rare cancer susceptibility syndromes have proved to have wide applicability in science in general and cancer in particular, such as Li-Fraumeni syndrome (p53), xeroderma pigmentosum (DNA nucleotide excision repair), and ataxia telangiectasia (P13 kinase). Fourth, basic work on FA has already led to clinical use of reagents for diagnosis and genetic counseling, and gene therapy trials are currently underway for treatment of FA. This work focuses on FA protein interactions. Preliminary data have shown that two FA proteins FANCA (Fanconi anemia complementation group A) and FANCC interact in a 500 kD nuclear complex, an interaction that is absent in 7 of the 8 FA complementation groups. Within the nucleus the data reveal that these proteins localize to the DNA and nuclear matrix-containing fractions and have an appearance that parallels that of other nuclear matrix proteins. Evidence is also presented for inducible localization to chromatin and for direct DNA binding on gel shift assay. The efforts in this proposal will be driven by and will directly test two hypotheses: l) that the FA nuclear complex functions as a multimeric complex whose isolation will yield additional binding partners which will enable us to clone FA complementation group genes, and 2) that the FA proteins are associated with the nuclear matrix and with DNA in a cell cycle-regulated, complementation group-dependent, and drug-inducible fashion and interact with DNA either directly or indirectly. First, the FA protein complex will be characterized with respect to size and inducibility. Second, The complex will be isolated and analyzed by mass spectroscopy in order to identify novel and known associated proteins. Third, the subcellular localization of the FA proteins and their interaction with DNA will be investigated in a number of ways, including those which can lead to further new protein isolation. Identification of new proteins and elucidation of FA pathway mechanisms will help uncover a new realm of cancer biology and directly provide clinical applicability.